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Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate. 1990

J E Reardon, and W H Miller
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.

3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was an efficient substrate for the human immunodeficiency virus 1 reverse transcriptase. It was incorporated into both homopolymer and defined sequence DNA-primed RNA templates and DNA-primed DNA templates. The substrate and inhibitor kinetics of both AZTTP and dTTP were dependent on the template-primer and reaction conditions used. dTMP was incorporated into poly(rA).oligo(dT) and into a defined sequence DNA-primed RNA template (when the other three 2'-deoxynucleoside 5'-triphosphates were present) as a conventional substrate, with steady-state Km values of 5-10 microM. The results suggest that the reverse transcriptase was capable of processive DNA polymerization on these DNA-primed RNA templates. In contrast, in the absence of the other three 2'-deoxynucleoside 5'-triphosphates, the time course for incorporation of dTMP into the same defined sequence DNA-primed RNA template was biphasic. A burst of product formation was observed followed by a slow steady-state rate with a Km value of 0.082 microM. AZTMP incorporation into poly(rA).oligo(dT) and into the defined sequence DNA-primed RNA template produced similar biphasic time courses and steady-state Km values. These results were consistent with rate-limiting dissociation of the polymerase.template-primer complex after "forced" termination of polymerization. AZTMP and dTMP were both incorporated into the homopolymer DNA-primed DNA template, poly(dA).oligo(dT), and a defined sequence DNA-primed DNA template as conventional substrates. Their Km values were similar (2-10 microM). The absence of biphasic time courses suggested that dissociation of the DNA-primed DNA templates from the enzyme, after forced termination, was not rate-limiting. This was consistent with a more distributive mode of DNA polymerization. With the defined sequence template-primers and poly(dA).oligo(dT), Ki values for both dTTP and AZTTP were comparable to their Km values. Thus, AZTTP appeared to be a simple competitive substrate-inhibitor with respect to dTTP. AZTTP inhibition of dTMP incorporation into poly(rA).oligo(dT) was linear competitive at low concentrations (0-100 nM) of AZTTP (Ki = 35 nM) but became hyperbolic (decreasing potency) at concentrations of AZTTP above this range. A mechanism for this nonlinear inhibition is discussed.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D000998 Antiviral Agents Agents used in the prophylaxis or therapy of VIRUS DISEASES. Some of the ways they may act include preventing viral replication by inhibiting viral DNA polymerase; binding to specific cell-surface receptors and inhibiting viral penetration or uncoating; inhibiting viral protein synthesis; or blocking late stages of virus assembly. Antiviral,Antiviral Agent,Antiviral Drug,Antivirals,Antiviral Drugs,Agent, Antiviral,Agents, Antiviral,Drug, Antiviral,Drugs, Antiviral
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012194 RNA-Directed DNA Polymerase An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49. DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA

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